Type 1 diabetes (T1D) is an autoimmune disorder with unambiguous involvement of both innate and adaptive immune mechanisms in the destruction of pancreatic beta cells. Recent evidence demonstrated that neutrophils infiltrate the pancreas prior to disease onset and therein extrude neutrophil extracellular traps (NETs), web-like structures of DNA and nuclear proteins with a strong pro-inflammatory biologic activity. Our previous work showed that T1D NETs activate dendritic cells, which consequently induce IFNγ-producing Th1 lymphocytes. The aim of this study was to assess direct ex vivo biomarkers of NETosis in the serum of recent onset and long-term pediatric T1D patients, their first-degree relatives and healthy controls. To this end we evaluated serum levels of myeloperoxidase (MPO), neutrophil elastase (NE), proteinase 3 (PR3), protein arginine deiminase 4 (PAD4), LL37 and cell-free DNA-histone complexes in sex- and age-matched cohorts of T1D first-degree relatives, recent-onset T1D patients, and in patients 12 months after clinical manifestation of the disease. Our data shows that disease onset is accompanied by peripheral neutrophilia and significant elevation of MPO, NE, PR3, PAD4 and cell-free DNA-histone complexes. Most biomarkers subsequently decrease but do not always normalize in long-term patients. First-degree relatives displayed an intermediate phenotype, except for remarkably high levels of LL37. Together, this report provides evidence for the presence of ongoing NETosis in pediatric patients with T1D at time of clinical manifestation of the disease, which partly subsides in subsequent years.

Department of Immunology 2nd Faculty of Medicine Charles University University Hospital in Motol Prague Czechia

Department of Pediatrics 2nd Faculty of Medicine Charles University and Motol University Hospital Prague Czechia

See more in PubMed

Valle A, Giamporcaro GM, Scavini M, Stabilini A, Grogan P, Bianconi E, et al. . Reduction of Circulating Neutrophils Precedes and Accompanies Type 1 Diabetes. Diabetes (2013) 62:2072–7.  10.2337/DB12-1345 PubMed DOI PMC

Vecchio F, Lo Buono N, Stabilini A, Nigi L, Dufort MJ, Geyer S, et al. . Abnormal Neutrophil Signature in the Blood and Pancreas of Presymptomatic and Symptomatic Type 1 Diabetes. JCI Insight (2018) 3:1–17.  10.1172/jci.insight.122146 PubMed DOI PMC

Harsunen M, Puff R, D’Orlando O, Giannopoulou E, Lachmann L, Beyerlein A, et al. . Reduced Blood Leukocyte and Neutrophil Numbers in the Pathogenesis of Type 1 Diabetes. Horm Metab Res (2013) 45:467–70.  10.1055/s-0032-1331226 PubMed DOI

Jackson MH, Collier A, Nicoll JJ, Muir AL, Dawes J, Clarke BF, et al. . Neutrophil Count and Activation in Vascular Disease. Scott Med J (1992) 37:41–3.  10.1177/003693309203700205 PubMed DOI

Yu Y, Lin Q, Ye D, Wang Y, He B, Li Y, et al. . Neutrophil Count as a Reliable Marker for Diabetic Kidney Disease in Autoimmune Diabetes. BMC Endocr Disord (2020) 20:158.  10.1186/s12902-020-00597-2 PubMed DOI PMC

Diana J, Simoni Y, Furio L, Beaudoin L, Agerberth B, Barrat F, et al. . Crosstalk Between Neutrophils, B-1a Cells and Plasmacytoid Dendritic Cells Initiates Autoimmune Diabetes. Nat Med (2012) 19:65–73.  10.1038/nm.3042 PubMed DOI

Brinkmann V, Reichard U, Goosmann C, Fauler B, Uhlemann Y, Weiss DS, et al. . Neutrophil Extracellular Traps Kill Bacteria. Science (2004) 303:1532–5.  10.1126/science.1092385 PubMed DOI

Wong SL, Demers M, Martinod K, Gallant M, Wang Y, Goldfine AB, et al. . Diabetes Primes Neutrophils to Undergo NETosis, Which Impairs Wound Healing. Nat Med (2015) 21:815–9.  10.1038/nm.3887 PubMed DOI PMC

Qin J, Fu S, Speake C, Greenbaum CJ, Odegard JM. Netosis-Associated Serum Biomarkers Are Reduced in Type 1 Diabetes in Association With Neutrophil Count. Clin Exp Immunol (2016) 184:318–22.  10.1111/cei.12783 PubMed DOI PMC

Parackova Z, Zentsova I, Vrabcova P, Klocperk A, Sumnik Z, Pruhova S, et al. . Neutrophil Extracellular Trap Induced Dendritic Cell Activation Leads to Th1 Polarization in Type 1 Diabetes. Front Immunol (2020) 11:661.  10.3389/fimmu.2020.00661 PubMed DOI PMC

Njeim R, Azar WS, Fares AH, Azar ST, Kassouf HK, Eid AA. Netosis Contributes to the Pathogenesis of Diabetes and Its Complications. J Mol Endocrinol (2020) 65:R65–76.  10.1530/JME-20-0128 PubMed DOI

Wang Y, Xiao Y, Zhong L, Ye D, Zhang J, Tu Y, et al. . Increased Neutrophil Elastase and Proteinase 3 and Augmented NETosis Are Closely Associated With β-Cell Autoimmunity in Patients With Type 1 Diabetes. Diabetes (2014) 63:4239–48.  10.2337/db14-0480 PubMed DOI

Klocperk A, Petruzelkova L, Pavlikova M, Rataj M, Kayserova J, Pruhova S, et al. . Changes in Innate and Adaptive Immunity Over the First Year After the Onset of Type 1 Diabetes. Acta Diabetol (2019) 57(3).  10.1007/s00592-019-01427-1 PubMed DOI

Petruzelkova L, Ananieva-Jordanova R, Vcelakova J, Vesely Z, Stechova K, Lebl J, et al. . The Dynamic Changes of Zinc Transporter 8 Autoantibodies in Czech Children From the Onset of Type 1 Diabetes Mellitus. Diabetes Med (2014) 31:165–71.  10.1111/dme.12308 PubMed DOI

Wang Y, Li M, Stadler S, Correll S, Li P, Wang D, et al. . Histone Hypercitrullination Mediates Chromatin Decondensation and Neutrophil Extracellular Trap Formation. J Cell Biol (2009) 184:205–13.  10.1083/jcb.200806072 PubMed DOI PMC

Nagaoka I, Suzuki K, Niyonsaba F, Tamura H, Hirata M. Modulation of Neutrophil Apoptosis by Antimicrobial Peptides. ISRN Microbiol (2012) 2012:1–12.  10.5402/2012/345791 PubMed DOI PMC

Zuo Y, Yalavarthi S, Shi H, Gockman K, Zuo M, Madison JA, et al. . Neutrophil Extracellular Traps in COVID-19. JCI Insight (2020) 5(11).  10.1172/JCI.INSIGHT.138999 PubMed DOI PMC

Fornasari PM. SARS-Cov-2 Protease-Dependent Human Cell Infection Induces Immunological Response Imbalance and Neutrophils “Proteolytic Storm”. Proteolysis Inhibitors Can Prevent and Treat Covid-19. J Cardiol Res Rev Rep (2021) 2:1–10.  10.47363/jcrrr/2021(2)135 DOI

De Beeck AO, Eizirik DL. Viral Infections in Type 1 Diabetes Mellitus-Why the β Cells? Nat Rev Endocrinol (2016) 12:263–73.  10.1038/nrendo.2016.30 PubMed DOI PMC

Increased histone citrullination in juvenile idiopathic arthritis